too high qPCR efficiency

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Petrusha
Petrusha's picture
too high qPCR efficiency

Hello,
when I´m running qPCR (standard is plasmid DNA with insert of our interest) with 5 standards (10-fold dilutions) I still have PCR efficiency around 160% or more (with Sybr and Probe as well)-slope around -2,55. 
Moreover I tried to delete some dilutions to overcome this but result is even worse. I didn´t use touchdown (what I´ve heard can slightly increase efficiency) and I know that only 1 product is amplified (according gel elfo and HRM).
I´ve tried different reactions (completely different inserts in the same plasmid) still having this too high efficiency and delta Ct is still around 2-2.5.
 Any explanation or suggestions?
Thanks

Ivan
Ivan's picture
 

 
Hi Petrusha,
A very high efficiency is typically due to an inhibitor being present in your DNA sample. This happens when your DNA was not fully cleaned; in other words, there are traces of proteins and other contaminants. A good way to think about this is that as you add more DNA to your qPCR reactions (higher dilutions), there is more inhibitor present and as a result you get less signal (higher Ct values) than the equivalent signal for the more dilute samples (which has proportionally less inhibitor). 
I suggest you get rid of the higher concentration dilutions and see what happens. I assume the Ct values for those are lower than 10-15. If you look at the difference in Ct values between your different dilutions my guess is that at higher DNA concentrations the difference between one dilution and the next is small (around -2) while at lower DNA concentrations the difference is higher (closer to -3.3)
Good luck

Petrusha
Petrusha's picture
Hello Ivan,

Hello Ivan,
thanks for advice, I did not think about DNA quality problem since Aratio was 1.8-2 and I relied on commercial isolation kit the lab uses usually for this purposes. I´ll try purify the sample and do it again.

Petrusha
Petrusha's picture
Helo Ivan,

Helo Ivan,
I purified my plasmid DNA and still have the same problem even I tried to get rid of the highest concentrated samples. I looked only for lower concentrations and the difference in Ct is still around -2.5. Aratio is around 2 so I guess it is not DNA quality problem.
Any suggestions?

Ivan
Ivan's picture
 

 
Hi Petrusha,
If you are sure that the problems is not the DNA, then it is likely the assay itself. I would suggest designing a new assay, and getting new primers, and running the qPCR again. Whenever I am designing a new assay I typically test at least 3 different primer combinations to make sure I get a very good assay.